Delivering the Goods for Genome Engineering and Editing

Abstract

A basic understanding of genome evolution and the life and impact of microorganisms, like viruses and bacteria, has been fundamental in the quest for efficient genetic therapies. The expanding tool box for genetic engineering now contains transposases, recombinases, and nucleases, all created from naturally occurring genome-modifying proteins. Whereas conventional gene therapies have sought to establish sustained expression of therapeutic genes, genomic tools are needed only in a short time window and should be delivered to cells ideally in a balanced "hit-and-run" fashion. Current state-of-the-art delivery strategies are based on intracellular production of protein from transfected plasmid DNA or in vitro-transcribed RNA, or from transduced viral templates. Here, we discuss advantages and challenges of intracellular production strategies and describe emerging approaches based on the direct delivery of protein either by transfer of recombinant protein or by lentiviral protein transduction. With focus on adapting viruses for protein delivery, we describe the concept of "all-in-one" lentiviral particles engineered to codeliver effector proteins and donor sequences for DNA transposition or homologous recombination. With optimized delivery methods-based on transferring DNA, RNA, or protein-it is no longer far-fetched that researchers in the field will indeed deliver the goods for somatic gene therapies.

Anemi tedavisinde gen düzenleme çalışmaları

“…Delivery methods of genome editing tools can be categorized into two, as viral and non-viral delivery. Considering non-viral delivery approach, cells can be transfected with plasmid DNA containing and expressing gene editing components, in vitro transcribed mRNA to induce translation of nuclease in host cell, or direct delivery of purified nucleases with donor repair templates (Skipper and Mikkelsen 2015). Donor template delivery is also versatile that it could be introduced to target cells in the form of plasmid DNA, dsDNA or ssDNA linear oligo (Chen, et al 2011;Orlando, et al 2010).…”

“…Combination of chemical supplements and transfection methods would result in an increase in efficiency but also toxicity and stress related cell death. Delivery of in vitro transcribed mRNAs encoding engineered nucleases (ZFN, TALEN or Cas9+gRNA) appears to be more advantageous due to lower genotoxicity and transient nuclease activity (Skipper and Mikkelsen 2015). Direct delivery of engineered nucleases as purified proteins using electroporation has been applied as another alternative approach to achieve temporary and safe genome editing .…”

“…Advances in genome engineering have brought in improvements in viral transfer tools and methods. Generation of non-integrating viral vector such as adenovirus vectors (AdVs), adeno-associated viral vectors (AAVs) and integrase deficient lentivirus vectors (IDLVs) enabled successful transfer of genes required for the expression of gene editing tool in host cells in both in vitro and in vivo (Skipper and Mikkelsen 2015). Each viral vector mentioned above possesses different characteristics in terms of transduction efficiency, packaging size and target cell type.…”

Gene Editing in Adult Hematopoietic Stem Cells

“…One of the most important steps to consider when performing a gene editing strategy is the delivery of the gene editing tools. In fact, nowadays this is probably the main bottleneck in gene therapy in general and definitely in the gene editing of HSCs (91).…”

“…Alternative platforms to deliver nucleases have been explored, such as the transfection of in vitro-transcribed mRNA (97,98). The main advantage is that mRNA establishes a short-term boost of enzyme activity with low toxicity and no risk of integration (99). Thus in vitro transcribed mRNAs that encode ZFNs, TALENs, or Cas9 and gRNAs are the preferential forms in which these nucleases are nucleofected into HSCs.…”